The amount of information concerning the genomes of a large variety of species is increasing exponentially. The availability of known sequences creates an enormous market for the detection of particular sequences present as DNA or RNA, whereby one can detect the presence of genes, their transcription or mutations, such as lesions, substitutions, deletions, translocations, and the like. By knowing sequences of interest, one can detect a wide variety of pathogens, particularly unicellular microorganisms and viral strains, and genetic diseases including the presence of genes imparting antibiotic resistance to the unicellular microorganisms, as illustrative of only a few of the available possibilities. In addition, there are needs within the extensive areas of genetic counseling, forensic medicine, research, and the like, for nucleic acid sequence detection technology.
In many instances, the target nucleic acid sequence is only a very small proportion of total nucleic acid in the sample. Furthermore, there may be many situations where the target nucleic acid of interest and other sequences present have substantial homology. It is therefore important to develop methods for the detection of the target nucleic acid sequence that are both sensitive and accurate.
Furthermore, oligonucleotide probe-based assay methods are known to depend upon careful optimization of the wash stringency. If the wash conditions are too stringent, then probe/target hybrids will be denatured, resulting in a decrease in the amount of signal in the assay. If the wash conditions are not sufficiently stringent, then non-specifically bound probes or mismatched probe/target hybrids will remain in the assay medium, resulting in high levels of non-specific or background signal in the assay. Optimal conditions are necessarily different for each probe because hybridization is a sequence-dependent phenomenon and would also depend on the extent to which near-homologous sequences are present in the sample.
The use of crosslinkable probes in nucleic acid hybridization assays to crosslink to target sequences is demonstrated in U.S. Pat. No. 4,826,967 by K. Yabusaki et al.; compounds are based on furocoumarin, (or psoralen) attached to existing polynucleotides (usually through adduct formation) and are satisfactory for many applications. However the crosslinking group/nucleoside adduct is difficult to synthesize particularly in large quantities. In U.S. Pat. No. 5,082,934, Saba et a. describe a photoactivatible nucleoside analogue comprising a coumarin moiety linked through its phenyl ring to the 1-position of a ribose or deoxyribose sugar moiety in the absence of an intervening base moiety. The resulting nucleoside analogue is used as a photo-crosslinking group when inserted into a polynucleotide as a replacement for one or more of the complementary nucleoside bases present in a probe used in hybridization assays. Nevertheless, new types of compounds that offer additional advantages, such as stability throughout probe synthesis and use, and conformational flexibility, continue to remain desirable.
There is, therefore, substantial interest in identifying alternative techniques which allow for the detection of specific DNA sequences and avoid the deficiencies of the other systems.